Replacement of feline foamy virus bet by feline immunodeficiency virus vif yields replicative virus with novel vaccine candidate potential
Hosts are able to restrict viral replication to contain virus spread before adaptive immunity is fully initiated. Many viruses have acquired genes directly counteracting intrinsic restriction mechanisms. This phenomenon has led to a co-evolutionary signature for both the virus and host which often provides a barrier against interspecies transmission events. Through different mechanisms of action, but with similar consequences, spumaviral feline foamy virus (FFV) Bet and lentiviral feline immunodeficiency virus (FIV) Vif counteract feline APOBEC3 (feA3) restriction factors that lead to hypermutation and degradation of retroviral DNA genomes. Here we examine the capacity of vif to substitute for bet function in a chimeric FFV to assess the transferability of anti-feA3 factors to allow viral replication.
We show that vif can replace bet to yield replication-competent chimeric foamy viruses. An in vitro selection screen revealed that an engineered Bet-Vif fusion protein yields suboptimal protection against feA3. After multiple passages through feA3-expressing cells, however, variants with optimized replication competence emerged. In these variants, Vif was expressed independently from an N-terminal Bet moiety and was stably maintained. Experimental infection of immunocompetent domestic cats with one of the functional chimeras resulted in seroconversion against the FFV backbone and the heterologous FIV Vif protein, but virus could not be detected unambiguously by PCR. Inoculation with chimeric virus followed by wild-type FFV revealed that repeated administration of FVs allowed superinfections with enhanced antiviral antibody production and detection of low level viral genomes, indicating that chimeric virus did not induce protective immunity against wild-type FFV.
Unrelated viral antagonists of feA3 cellular restriction factors can be exchanged in FFV, resulting in replication competence in vitro that was attenuated in vivo. Bet therefore may have additional functions other than A3 antagonism that are essential for successful in vivo replication. Immune reactivity was mounted against the heterologous Vif protein. We conclude that Vif-expressing FV vaccine vectors may be an attractive tool to prevent or modulate lentivirus infections with the potential option to induce immunity against additional lentivirus antigens.
KeywordsFoamy virus In vitro virus evolution In vivo host factor requirement Replicating vaccine vector Lentivirus Bet function Vif function APOBEC3 Restriction factor Superinfection
feline foamy virus
feline immunodeficiency virus
human immunodeficiency virus
feline A3 proteins
wild-type pCF-7 inoculated group
chimeric pCF7-Vif W/*1 inoculated group
naïve control group
peripheral blood mononuclear cell(s)
Colorado State University
multiplicity of infection
Crandell feline kidney cell(s)
human embryonic kidney cell(s)
weight per volume
Foamy viruses (FVs) are ancient retroviruses comprising the only genus of the subfamily Spumaretrovirinae, which are different in many aspects from the Orthoretrovirinae that comprise all other known retroviruses including lentiviruses (LVs) [1, 2, 3]. Despite having a wide tissue tropism in infected animals, FVs have historically been regarded as apathogenic and are endemic in primates, bovids, felids, and other hosts. Clusters of highly related viruses have been documented in closely related hosts [4, 5, 6, 7]. While humans do not have endemic FVs, they are susceptible to zoonotic infections from non-human primates [8, 9]. FVs and LVs such as feline immunodeficiency virus (FIV) have been used to develop vectors for vaccine antigen delivery and gene therapy in a variety of mammals [10, 11, 12, 13, 14, 15, 16, 17]. In domestic cats (Felis catus), feline foamy virus (FFV) and FIV establish lifelong infections despite specific host antiviral immune responses [18, 19, 20, 21]. In contrast to FFV infection, FIV infection leads to the development of an immunosuppressive AIDS-like syndrome in some cats [18, 20, 22, 23, 24]. Thus, FVs are an attractive alternative to LV vectors due to their apathogenicity, wide tissue tropism, and establishment of a persistent infection with ongoing virus gene expression and replication [6, 7, 12, 13, 21, 25, 26]. Other advantageous features of FV-based vectors are a safer integration profile than gammaretroviral and LV vectors [11, 27], a large packaging capacity, and the ability to introduce self-inactivating properties [17, 28, 29, 30, 31]. Investigating FV vector candidates could thus yield potential new therapies to benefit both humans and animals .
Both LVs and FVs are complex retroviruses encoding the canonical Gag, Pol, and Env proteins, regulatory proteins essential for replication in all cells, and accessory proteins required only in certain cells. For instance, LV Tat and FV Tas (also designated Bel1) proteins are both transactivators for virus gene expression, however, their mode of action is completely different (for review ). Regardless, both regulatory genes induce a positive feedback loop to generate more transactivator protein in addition to transcription of structural genes required for infectivity . FVs additionally encode Bet that is generated via splicing, consisting of N-terminal Tas sequences while the majority of the protein is encoded by another reading frame, the bel2 gene . Bet is the functional homologue of the LV Vif protein, both of which are involved in countering the host intrinsic antiviral restriction factors of the APOBEC3 (A3) family [33, 34, 35, 36, 37, 38].
Like all other viruses, LVs and FVs are restricted by intrinsic cell mechanisms that impair or even suppress the different phases of virus replication, progeny production, and establishment of infection in the new host (for review see [39, 40]). Nonspecific innate immunity and cell-based intrinsic immunity employing antiviral restriction factors are both absolutely required to control pathogen replication before adaptive immunity matures for long-term suppression of viral replication [41, 42]. Therefore, a fine-tuned crosstalk between innate, intrinsic, and adaptive immunity is needed to control and eliminate the pathogen as well as to build up immunological memory [41, 42, 43]. Pathogens have evolved a plethora of counteracting strategies in order to evade this control, often by the acquisition of counteracting proteins [39, 40]. The idea and concept of host-encoded restriction factors and the viral counter-defense have been in part established in human immunodeficiency virus (HIV) research. These initial studies analyzed the interplay between host-encoded A3 cytidine deaminases that result mainly in lethal mutagenesis (C to U/T exchanges) of the retroviral HIV genome during reverse transcription, and the counter-defense by LV Vif (or Bet in FVs) which result in A3 degradation (via Vif) or sequestration (via Bet) [33, 34, 36, 40, 44].
Analogous to human A3 function, feline A3 (feA3) proteins are produced in many cell types and introduce missense and stop mutations into nascent viral genomes, ultimately restricting viral replication through hypermutation and degradation [33, 34, 39]. Several studies on the function of FIV Vif and FFV Bet, which are of very different size and share no obvious sequence or structural homology [36, 38, 45], have revealed that they employ completely different modes of action to achieve the same end goal: preventing the packaging of feA3 proteins into the particle to avoid subsequent viral lethal mutagenesis. The FIV Vif protein (25 kDa) functions as an adapter molecule, binding to cognate or highly-related feA3 proteins and recruiting the ubiquitin proteasome degradation machinery, resulting in the removal of feA3 proteins from the virus-producing cell [44, 46, 47, 48, 49]. This is the critical prerequisite to prevent cytidine deamination during or after reverse transcription of the genome. In contrast, FV Bet proteins (of 43 to 56 kDa) tightly bind A3 proteins of their cognate host species without leading to degradation, likely acting via sequestration or blocking of essential binding and multimerization sites [34, 36, 37, 45]. Therefore, vif and bet are essential viral genes required to allow productive replication in cells with active A3 expression [39, 50, 51].
Domestic cats produce multiple A3 proteins in one and two-domain forms. One-domain feA3 proteins include the A3Z2 (present as A3Z2a, A3Z2b, and A3Z2c) and A3Z3 isoforms, while read-through transcription leads to the production of two-domain feA3Z2-Z3 proteins (in A3Z2b-Z3 and A3Z2c-Z3 isoforms) . These feA3 proteins have differential effects on FFV and FIV: A3Z2s markedly reduce titers of FFV lacking bet, while the A3Z3 and A3Z2-Z3 proteins inhibit FIV virions lacking vif with intermediate and high efficiency, respectively. Interestingly, both Bet and Vif counteract all feA3 regardless of whether the specific A3 isoforms efficiently restrict FFV or FIV [33, 34, 44, 46, 47, 52], suggesting a more complex relationship between these accessory genes and host restriction factor regulation than has yet been described.
Here we describe the generation and in vitro selection of FFV-Vif chimeras in which FIV vif partially or almost fully restored the replication capacity of bet-deficient FFV constructs in vitro. An in vitro-selected FFV-Vif variant that drives expression of the heterologous lentivirus Vif independent from any FFV protein and which is highly dependent on Vif expression in A3-producing cells, was used for infection of domestic cats to test the chimera’s replication competence and immunogenicity. Replication of the FFV-Vif chimera was attenuated in cats compared to wild-type FFV. Cats infected with the FFV-Vif chimera developed persistent antibody responses towards FFV proteins and FIV Vif but proviral FFV-Vif chimeric genomes were at or below the limit of detection in peripheral blood mononuclear cells (PBMC) of infected cats. In contrast, proviral genomes were consistently detected in wild-type FFV-infected cats. Inoculation of cats in the FFV-Vif chimera cohort with wild-type FFV or re-inoculation with FFV-Vif chimeric virus boosted anti-FFV Gag antibody titer following re-infection. These results suggest that compensatory changes arising in vitro seemingly allowed FIV-Vif to substitute for FFV-Bet function, but were incapable of fully supporting FFV-Vif chimeric replication competence in vivo. These findings additionally suggest the capacity of spumaviruses to superinfect cats following prior attenuated FFV replication, indicating the potential suitability of chimeric FFV as a vaccine vector in the face of a pre-existing infection and immunity.
FIV Vif and FFV Bet confer protection from feA3 restriction in vitro
Previous studies have shown that the FIV Vif accessory protein has the capacity to direct proteasomal degradation of all known feA3 cytidine deaminase restriction factors irrespective of whether they strongly or moderately restrict FIV replication [44, 46, 47, 52]. Similarly, FFV Bet binds to all feA3 isoforms and inactivates their restriction potential by a degradation-independent, different mechanism not comparable to FIV Vif [33, 34]. In addition, FIV Vif can protect the replication capacity of bet-deficient FFV while FFV Bet correspondingly counteracts feA3-mediated restriction of vif-deficient FIV [39, 44].
Viral clones and stocks used in this study
Viral stock namea
Effect on replication (CrFK)
Truncation at Bet amino acid 117
Fully susceptible towards feA3-mediated restriction in vitro
Insertion and replacement of Bet residues at amino acid 117 by insertion of a multiple cloning site
Fully susceptible towards feA3-mediated restriction in vitro
Engineered Bet-Vif fusion protein
Partially susceptible towards feA3-mediated restriction in vitro
Fully susceptible towards feA3-mediated restriction in vitro
Trp to Stop mutation (TGG to TGA), unlinked vif gene
Enhanced, compared to pCF7-Vif-4
Trp to Stop mutation (TGGG to TAGA), unlinked vif gene
Enhanced, compared to pCF7-Vif-4
pCF7-Vif W/*1 M+
Optimized upstream Met codon in pCF7-Vif W/*1
Similar to pCF7-Vif W/*1
pCF7-Vif W/*2 M+
Optimized upstream Met codon in pCF7-Vif W/*2
Similar to pCF7-Vif W/*2
pCF7-Vif W/*1 M/T
Upstream Met codon mutated to Thr in pCF7-Vif W/*1
Similar to pCF7-Vif W/*1
pCF7-Vif W/*2 M/T
Upstream Met codon mutated to Thr in pCF7-Vif W/*2
Similar to pCF7-Vif W/*2
Substitution of FFV Bet by functional Vif confers FFV replication competence in feA3 expressing cells
Passage through CrFK enhances FFV-Vif chimera replication efficiency
We continued passaging progeny of wild-type pCF-7 and chimeric pCF7-Vif-4 (see above, Fig. 2a) for 20 passages in order to use in vitro selection and evolution to obtain FFV-Vif variants with higher replication capacity in the presence of the feA3 proteins endogenously expressed in CrFK cells . During the first seven passages, wild-type pCF-7 displayed titers between 106 and 107 focus-forming units per ml (FFU/ml) (Fig. 2a). During this phase, infectivity of the chimeric clone pCF7-Vif-4 was approximately one to two logs lower (104–106 FFU/ml). Starting at passage eight, however, titers of pCF7-Vif-4 progeny approached that of wild-type pCF-7, indicating emergence of pCF7-Vif variants with enhanced replicative ability in vitro (Fig. 2a). Selected samples harvested during CrFK passaging were analyzed for FFV Gag and Vif expression (Fig. 2b). FFV Gag expression was consistently detectable in all cell lysates using FFV reference serum from cat 8014 (Fig. 2b, middle panel). Early, during viral passages 2 and 5, the BettrVif fusion protein and its proteolytic cleavage products were the primary Vif-reactive proteins detectable. At passage 10, BettrVif became undetectable and the Vif protein of approximately 25 kDa was detected, along with additional Vif-reactive bands of higher molecular mass. At passage 15, mostly Vif proteins in the 25 kDa size range were identified (Fig. 2b, top panel).
To detect potential adaptive genetic changes in the FFV genome, DNA was prepared from FFV-Vif-4-infected CrFK cells at passage 18 and used as template for PCR to amplify and clone the complete bet tr vif region. In seven of nine amplicons, a tryptophan codon (TGG, Trp) located in the bet sequence 50 codons upstream of the vif ORF had mutated to become TAG and TGA stop codons (Fig. 2c and Additional file 2). These changes were transitions of either the first or second G residue to an A (Fig. 2c, top panel) yielding two different stop codons, indicated by an asterisk (*), as either a TGA (five out of seven sequences, designated W/*1) or a TAG stop codon (two out of seven sequences, designated W/*2). In the five clones that had incorporated the TGA stop codon in the bet sequence, a G172R mutation in the overlapping Tas-coding sequence occurred. In the two TGG to TAG mutants, a G residue following the TGG codon was also changed to A (i.e. TGGG was altered to TAGA). This resulted in a G172L exchange in Tas and a D/N change directly downstream of the new bet stop codon. All nucleotide exchanges correspond to C/T exchanges of the antisense strand in a sequence context PyPyC (Fig. 2c; Py = pyrimidine residue), corresponding to the canonical A3 mutation context in retroviral genomes [34, 53]. Additional genetic changes were not consistently detected in the bet tr vif region.
Unlinking vif from bet by Trp/stop mutagenesis is essential for increased infectivity
The importance of the identified Trp/stop (W/*) mutations upstream of the vif sequence was analyzed using reverse genetics. Both W/* mutations in the bel2 linker sequence upstream of vif were inserted into the original pCF7-Vif-4 to determine whether they represent adaptive mutations increasing the titer of the corresponding FFV-Vif chimera. These clones were named pCF7-Vif W/*1 (TGG/TGA) and pCF7-Vif W/*2 (TGG/TAG, Table 1).
An additional outcome of the W/* mutations was the “emergence” of an in-frame ATG codon between the new W/* stop codon and the authentic vif start codon (Additional file 2). To test whether this ATG codon could serve as an alternative translational initiation codon for the inserted vif gene, this Met ATG was replaced in the engineered pCF7-Vif W/*1 and -W/*2 clones and the parental pCF7-Vif-4 clone by a threonine (Thr) codon (suffix M/T, see Fig. 2c lower panel and Table 1). In addition, and as a complementing strategy, the surrounding nucleotide sequence of this ATG codon was converted to an optimal Kozak translational initiation context sequence (GCCA/GCCATGG, start codon underlined, ) as shown in Fig. 2c, lower panels. The corresponding clones are labeled by the suffix M + (Table 1). The M/T mutation resulted in a silent mutation at the tas C-terminus while the change to a Kozak sequence resulted in two amino acid exchanges in tas at the C-terminus, i.e. D206H and A208G, and, in addition, a leucine to phenylalanine (L/F) exchange upstream, and a leucine to valine (L/V) exchange directly downstream of the potential Met start codon in the linker sequence (see Fig. 2c).
Transient co-transfection studies using a luc FFV LTR reporter construct together with either a CMV-IE promoter-driven Tas expression clone and a CMV-IE-driven β-gal plasmid or the FFV genomes pCF-7, pCF7-Vif-4, pCF7-Vif W/*1, and the different M/T and M + derivatives thereof were conducted. While the CMV-IE promoter-driven Tas expression clone yielded very high luc activities, the genomic wild-type and chimeric proviral FFV clones described above did not show significant differences in Tas transactivation, indicating that the mutations introduced do not significantly influence overall transactivation and gene expression (Additional file 3).
Clones pCF7-Vif W/*1 and -W/*2 and the different M/T and M + derivatives were transfected into HEK 293T cells and supernatants were tested for the replication competence of the FFV-Vif chimera in feA3-positive CrFK cells by serial CrFK cell passaging as described above. Serial passaging after either 60 or 84 h (Additional file 4A and 4B) showed similar outcomes: the pCF-7-encoded wild-type FFV had slightly higher titers (about fivefold) than mutants pCF7-Vif W/*1 and -W/*2 and their derivatives. For these clones and the corresponding M/T and M + clones, titers were stable during serial passages. This was not the case for the original pCF7-Vif-4 clone encoding the BettrVif fusion protein, where titers steadily and reproducibly declined upon serial passages in several independent experiments. The data show that both W/* mutations in the FFV bet sequence upstream of the vif gene cause in feA3-expressing CrFK cells a clear increase of replication competence compared to the pCF-Vif-4 encoding the BettrVif fusion protein. However, the replication competence of the pCF7-Vif W/*1 and -W/*2 clones was slightly lower than that of the wild-type FFV genome pCF-7. In addition, the FFV-encoded, in-frame ATG codon located 14 codons upstream of vif is probably not used as a start codon for Vif protein expression since its replacement by a Thr codon, or the optimization of the surrounding residues towards more efficient translational initiation, did not significantly affect viral titers.
Reduced steady state levels of feA3Z2b by FFV-Vif chimeric clones pCF-Vif-4 and pCF7-Vif W/*1 and -W/*2
Experimental infection of cats with chimeric virus FFV-Vif W/*1
Wild-type inoculated cats exhibited persistent FFV DNA proviral loads in PBMC in contrast to chimera-inoculated cats
Three out of four cats inoculated with only FFV-Vif chimeric virus (CH group) showed indeterminate results for FFV PBMC provirus DNA by qPCR analysis at some of the time points tested prior to re-inoculation on day 53 p.i. (Fig. 5b and Additional file 5). One of the chimera-inoculated cats re-inoculated on day 53 with wild-type virus (cat CH2WT) demonstrated FFV proviral DNA in PBMC 24 d post re-inoculation, while the other cat in this cohort remained indeterminate or negative throughout the study (Fig. 5c). The highest viral load recorded for cat CH2WT was 656 viral copies/106 cells 24 days post re-inoculation (Fig. 6). Both cats re-exposed to the FFV-Vif chimera displayed repeated indeterminate PCR results in blood before and after superinfection (Fig. 5b, d).
Gag-specific immune reactivity in infected animals confirms replication competence of wild-type FFV and FFV-Vif chimera
Infected cats seroconverted against accessory FFV Bet and FIV Vif proteins
This study describes the generation of replication-competent variants of FFV that express FIV Vif in lieu of FFV Bet. An engineered FFV genome expressing a fusion protein of a non-functional N-terminal Bet domain fused to the full-length Vif was clearly attenuated in vitro. Second-generation FFV-Vif chimeras expressing the authentic codon-optimized vif gene showed much higher vif-dependent replication competence in feA3-expressing cells, only slightly decreased in vitro compared to wild-type FFV. In experimentally infected cats, replication of the chimeric FFV-Vif variant was attenuated but led to the induction of FFV Gag-specific antibodies together with those directed against the engineered heterologous FIV Vif protein. Importantly, cats infected with the FFV-Vif chimera could be superinfected with wild-type FFV or the chimera, in both cases resulting in a strong immunological boost of sero-reactivity against FFV and FIV Vif.
The successful replacement of FFV bet by FIV vif in the context of the FFV genome may have been aided by two mechanisms. First, a codon-optimized and thus Rev-independent FIV vif gene was inserted, allowing for efficient translation of the Vif protein . Second, LV Vif proteins function as catalytic regulators of proteasomal feA3 degradation; therefore, much lower amounts of fully functional Vif may be required to inactivate A3 activity compared to FV Bet, which acts stoichiometrically via direct binding to the feA3 protein . Thus, the attenuated replication of the initially constructed pCF7-Vif-4 chimera was likely due to high expression levels of the functionally impaired BettrVif fusion protein.
In support of the hypothesis that Bet and Vif are differentially expressed in vivo, Bet sero-reactivity is high and has diagnostic value in infected cats and bovines [55, 56]. In contrast, while anti-Vif antibodies have been described in HIV patients [57, 58], Vif has not been shown to be a major humoral immune target of FIV infection, and seroconversion against Vif has not been well studied in FIV infection (personal communication, Dr. Chris Grant).
Apparently, inhibitory effects of either the complete N-terminal part of Bet plus the linker sequence, or the N-terminal residues of the linker residues present downstream of the engineered FFV PR cleavage site (see Fig. 1a) favored the emergence of Trp/stop (W/*) variants. This is strongly suggested by the fact that two independent, yet highly related mutational events led to the W/* mutation in the linker sequence upstream of vif. The reverse genetic experiments conducted do not support translational initiation at the upstream Met residue located in the linker sequence as important for Vif protein expression. We thus assume that in the in vitro-selected clones, fully functional Vif is expressed from its authentic start codon, though the exact mechanism by which FIV Vif protein is expressed from pCF7-Vif W/*1 and pCF7-Vif W/*2 is unknown. We assume that internal re-initiation of protein biosynthesis may be involved, but other mechanisms cannot be excluded. While the mechanism of Vif expression of clones pCF7-Vif W/*1 and pCF7-Vif W/*2 is unknown, all FIV Vif proteins engineered into the FFV genome including the first-generation clone pCF7-Vif-4 encoding the BettrVif fusion protein lead to dramatically reduced steady state levels of feA3 proteins as shown for the major restriction factor of FFV, feA3Z2b (see Fig. 3).
In line with the assumption that the original BettrVif fusion protein conferred suboptimal protection against feA3 restriction, both mutations leading to the adaptive W/* mutations occurred in a sequence context of the negative strand that is indicative of feA3 editing, suggesting the chimeric viruses did not confer robust protection against feA3. Both mutated C residues of the negative strand are preceded by C residues in the sequence 5’-TCCC-3’ (deaminated C residues in bold face letters, see also Fig. 2c), and therefore should function as optimal feA3 substrates, however, alternative mutational pathways might have also played a role. The fact that suboptimal feA3 inhibition leads to adaptive changes induced by feA3 DNA deamination supports our proposed concept that the heterologous and functionally relevant transgene FIV vif is essential for efficient propagation of the replicating virus, and thus confers a strong selective advantage by protecting against feA3 restriction. Consequently, the transgene vif has to be stably maintained in the absence of bet during serial passages, as demonstrated in Fig. 2b. The importance of the vif transgene for FFV-Vif replication is further underscored by the fact that additional adaptive changes, such as unlinking from N-terminal Bet sequences, were required to restore full biological activity as an inhibitor of feA3 restriction.
The advantage of adapting this replication-competent FV vector system as a vaccine delivery vehicle is that the immunogen Vif is essential for replication, and should be thus stably maintained by the engineered vector. Further, LV Vif has been shown to elicit T and B cell reactivity in HIV-infected individuals [59, 60, 61, 62, 63]. A corresponding PFV-based replicating vector system carrying the HIV vif gene may therefore be an interesting vector for the development of anti-HIV immunotherapies.
The in vivo wild-type FFV inoculations confirm that experimental infection of outbred, immunocompetent cats with clone pCF-7-derived wild-type FFV leads to a persistent infection with consistent detection of FFV proviral DNA in PBMC and a strong sero-reactivity against Gag and Bet proteins, similar to other reports [12, 21]. In contrast, animals inoculated with FFV-Vif W/*1 remained either proviral DNA negative or indeterminate throughout the study based on nested and qPCR analysis of PBMC DNA (Fig. 5b, d). Surprisingly, despite the inability to unambiguously detect FFV provirus in cats exposed to FFV-Vif W/*1, clear sero-reactivity against FFV Gag and heterologous Vif protein were detected after primary inoculation (Figs. 5, 7). This observation is consistent with previous studies in different FVs that serology is much more sensitive for the identification of exposed animals than PCR-based studies using PBMC [21, 55, 64, 65].
FFV in vivo infection experiments were conducted with wild-type FFV or chimeric FFV-Vif W/*1. This resulted in detectable, but low, proviral load in wild-type-infected animals and either undetectable or indeterminate proviral loads in cats infected with the FFV-Vif chimera. It is feasible that the exchange of Bet for Vif altered tissue tropism and site of viral replication in FFV-Vif W/*1 exposed cats, and this contributed to the inability of tracking viral infection via peripheral blood PCR. While initially either negative or indeterminate based on PCR results, cat CH2WT was superinfected with wild-type FFV on day 53 p.i. and showed a productive PBMC FFV infection on a similar timeline after inoculation as the wild-type-infected animals.
The animals in the chimeric cohort did not seroconvert against Bet as anticipated but they displayed clear anti-Vif antibody responses starting at day 15 p.i., demonstrating that substituting Bet by Vif elicited specific immune responses. Given that anti-Vif antibodies have not been widely reported during FIV infection, our findings may indicate replication is occurring in cells or cell compartments where it is more easily recognized as a foreign antigen. Antibody production against Vif was initially not robust, but following re-inoculation with both wild-type and FFV-Vif chimeric virus, anti-Vif antibody response markedly increased (Fig. 8b). Following re-inoculation of the FFV-Vif-infected animals with wild-type FFV virus, both cats initially infected with FFV-Vif W/*1 also produced anti-Bet antibodies, demonstrating that infection with the chimera did not protect against subsequent infection with wild-type FFV (Figs. 5c, 8a).
The data document that, despite a lack of consistent detection of FFV provirus DNA, FFV-Vif W/*1 is able to induce persistent antibody responses in domestic cats that were boosted by re-inoculation. As noted above, we were able to document superinfection with two highly related FFV variants. Clinical evaluations following exposure to wild-type FFV or chimera suggests that FFV produces an apathogenic infection during the study period. Further studies should be conducted to understand chronic FFV infection and potential associated pathologic changes as well as the possibility of worsened pathology following superinfection with other FFV serotypes or other viral infections . It would also be important to challenge wild-type FFV-infected cats with chimeric virus to determine whether infection with wild-type FFV may induce neutralizing immunity thus preventing superinfection with an attenuated FFV vaccine construct.
It cannot be ruled out that the antibody response detected in FFV chimera-infected animals was related to exposure to viral inoculum versus actively replicating virus, since PCR results were indeterminate. However, anti-Vif antibody production in three out of four FFV-Vif chimera-inoculated cats detected throughout the monitoring period, and an anti-Gag response equivalent to wild-type antibody titers is supportive of the conclusion that low-level viral replication occurred [21, 55, 64, 65]. Whether or not FFV-Vif W/*1 replicated poorly or not at all, the fact that pCF7-Vif W/*1 was highly replication-competent in CrFK cells but strongly attenuated in vivo suggests that Bet may play a currently unknown critical role in viral replication competence in vivo in addition to antagonizing A3-mediated restriction. Here, inactivation of other components of the host’s innate or intrinsic immunity as well as an essential co-factorial role for the replication in specific cell types in vivo are plausible reasons for the attenuated phenotype. Alternatively, other aspects of the manipulated pCF7-Vif W/*1 genome may impede replication in the native host. Further studies may elucidate additional complex host-virus restriction pathways that are relevant in vivo but are functionally masked or not relevant during in vitro infections.
Findings presented here illustrate a role for pCF7-Vif W/*1 to be used as a novel anti-LV vaccine delivery scaffold. This system would exploit a non-pathogenic vector that has to stably retain the Vif vaccine antigen and may be a therapeutic option to boost immunity towards an existing HIV infection in order to eliminate infected cells. The option to insert additional B and T cell epitopes at the terminus of the truncated Bet may be a means to extend and direct the host immune response towards additional epitopes (Slavkovic Lukic and Löchelt, unpublished observations). The ability to administer repeatedly or simultaneously the FV-based vaccine vector, directing expression of additional or newly acquired antigens, is an additional strength of our system as low level or absence of replication would hinder use of pCF7-Vif W/*1 as a vector delivery system that requires greater viral replication. Our results suggest that prior infection with wild-type FFV might not impair response to FFV-Vif, though superinfection studies will need to be conducted before this vector could be commercially developed. Experiments determining the viability of FV-LV Vif chimeric variants would also have to include assays to determine stability and functionality of inserted heterologous epitopes. Since we have documented that seroconversion occurs against Vif and Gag during FFV-Vif W/*1 exposure in the absence of intentional adjuvation, the attenuated replication does not impair its use as an antigen expression platform for eliciting antibodies against foreign antigens, and could even improve its biological safety.
Our in vitro and in vivo studies show the feasibility of constructing a replicative FFV-Vif vector that incorporates FIV Vif and replaces FFV Bet protein expression to counteract intrinsic feline A3 restriction factors. The FFV-Vif chimera inoculation of domestic cats induced a specific immune response against the heterologous Vif protein which under superinfection boosted antibody production against both FFV Gag and FIV Vif. Superinfection was also possible using wild-type FFV as evidenced by seroconversion against FFV Bet in animals initially inoculated with the chimeric construct, which provides plausibility of using this vector in domestic cat populations which may already be infected with wild-type virus. These findings demonstrate that this and additional FV vector systems may be further studied to develop potential therapeutic or preventive avenues against lentiviral infections including HIV.
Cells, culture conditions, and DNA transfection
Crandell feline kidney (CrFK) cells were used for FFV infection and propagation [34, 66, 67]. Human embryonic kidney (HEK) 293T cells used for plasmid transfection were propagated as described . FeFAB cells (CrFK-derived cells that carry a ß-galactosidase gene under the control of the FFV LTR promoter that is activated via FFV infection and subsequent Tas expression) were used to determine viral titer as described previously . PBMC were purified from feline blood using Histopaque gradients (Sigma Aldrich, St. Louis, MO). HEK 293T cells were transfected or co-transfected by using a modified calcium phosphate method described previously . In serial passage experiments, wild-type pCF-7 and Vif-chimera pCF-Vif-4 were transfected into HEK 293T cells . Supernatants were harvested 2 days post transfection and used to infect feA3-positive CrFK cells. Supernatants from these infections were serially passaged twice a week (every third or fourth day p.i.) to new, uninfected CrFK cells. A total of 20 serial passages were conducted.
FFV propagation and titration
For viral propagation of wild-type FFV and chimera (generated by transfection of HEK 293T cells), 106 CrFK cells/ml were seeded and infected at a multiplicity of infection (MOI) of 0.1. Supernatants were harvested and used for viral titer estimation and further viral propagation. FFV titers were determined using 5 × 104 FeFAB cells/well grown in 24-well plates and infected with serial 1:5 dilutions as described . Titers were calculated by determining the highest dilution that contained blue-colored infected cells through light microscopy.
Wild-type and FFV-Vif chimera viral propagation and titration for cat infections
2 µg of FFV pCF-7  or pCF7-Vif W/*1 plasmid were transfected into CrFK cells using Lipofectamine and supernatants were harvested for amplification in CrFK cells. Microscopic observation of cells was conducted daily and considered to be infected if they displayed cytopathic effects (CPE) of vacuolization, cytomegaly, and multinucleation [69, 70, 71]. Supernatants of infected cells were harvested and frozen on 2, 6, 9, and 13 days p.i. CPE end-point dilution titration was conducted on CrFK cells to determine TCID50/ml. CrFK (3 × 104 cells/well) were incubated with 25 µl of virus-containing supernatants in five-fold dilutions from the aforementioned days and observed for CPE up to 17 days p.i. The number of CPE-positive wells was used to determine TCID50/ml using the method of Reed and Muench . Supernatants that yielded the highest titers were selected for animal inoculations.
FIV titration system and FFV LTR luc reporter assay
Production of FIV luc reporter viruses, normalization according to reverse transcriptase activity, and target cell infection and reporter readout were done as previously described . FFV reporter assays using co-transfection of HEK 293T cells with the full-length FFV LTR luc reporter plasmid pFeFV-LTR-luc and the different FFV chimeras generated in this study or the FFV Tas expression construct pFeFV-Bel1 were conducted as described previously .
Primers used for cloning and PCR detection
Mutagenesis of the upstream start codon
PCR amplification of in vitro-selected FFV-Vif variants
Nested FFV PCR
Real-time quantitative PCR
Cloning and sequencing of in vitro selected FFV-Vif variants
DNA from CrFK cells infected with in vitro selected variants of pCF7-Vif-4 was harvested at passage 18 using the DNeasy extraction kit as specified by the supplier (Qiagen, Hilden, Germany). Sense primer FFV 9366 and antisense primer 10288 (Table 2) were used to amplify a 923 nt fragment of the bel1–vif region. Amplicons were cloned into pCR-TOPO TA vectors (Invitrogen, Karlsruhe, Germany) and subjected to in-house Sanger DNA sequencing of both strands.
Animals and experimental design
Twelve specific-pathogen-free (SPF) cats, aged 6–8 months and negative for common feline pathogens including FFV and FIV, were obtained from the Colorado State University (CSU) SPF Colony and housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited animal facility at CSU. All procedures were approved by the CSU Institutional Animal Care and Use Committee prior to initiation of the study. Cats were separated into three groups (n = 4 per group) based on inoculation type: FFV-negative CrFK culture media (naïve, N), wild-type FFV (WT), or chimeric FFV-Vif W/*1 (CH) (Fig. 4). Virus-inoculated animals received 105 TCID50 in 2 ml under ketamine anesthesia, split into 1 ml intramuscularly (i.m.) and 1 ml intravenously (i.v.). Cats were monitored daily for clinical signs of disease, and body temperature and weight were measured weekly. Peripheral blood was collected via cephalic or jugular venipuncture and processed to obtain serum and PBMC. On day 53 p.i., all cats in the CH cohort were re-inoculated each with 5 ml of undiluted virus (wild-type virus 2.78 × 105 TCID50/ml or chimeric virus 5.56 × 104 TCID50/ml, split into 1 ml i.m., 2 ml i.v., and 2 ml subcutaneously). Two of these cats were re-inoculated with wild-type FFV virus (henceforth referred to as CH1WT and CH2WT) and the other two cats with FFV-Vif W/*1 (now referred to as CH3CH and CH4CH). Animals were humanely euthanized for necropsy on day 176 p.i. (Fig. 4).
Nested and real-time quantitative PCR assays
Nested FFV PCR (nPCR) was performed on PBMC DNA to screen for initial infection status. Proviral DNA was purified and amplified using 0.5 µM gag-specific forward and reverse primers listed in Table 2 under the following cycling conditions for the first round of nPCR: 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min, and a final elongation step at 72 °C for 5 min. For the second round, 2 µl of first-round product was added to the reaction and amplified in these conditions: 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. Products were electrophoresed in 1.5% agarose gel in Tris-acetate buffer and stained with GelRed™ Nucleic Acid Gel Stain (Biotium, Hayward, CA) then visualized to look for the 333 base-pair PCR product. Real-time quantitative PCR (qPCR) was performed in triplicate on viral DNA as previously described  using 0.5 uM forward and reverse Gag-based primers and 0.1 uM probe (Table 2) with the following modified conditions: 95 °C for 3 min, 45 cycles of 95 °C for 10 s, and 60 °C for 40 s. Viral copy number quantification was based on a plasmid standard curve prepared from plasmid pCF-7. FFV-Gag real time PCR assay sensitivity is 1–10 viral copies per reaction . Infection status was divided into 3 categories: positive, negative, and indeterminate. Animals considered unequivocally “positive” had qPCR results with Cq values less than or equal to 37 in 2–3 out of the three reactions, consistent with viral load greater than 10 copies/reaction. Animals considered “negative” were negative for all triplicate tests (this included all naïve cats and “no template” controls at all defined times). Animals classified as “indeterminate” had qPCR replicates with Cq values > 37, equivalent to 0–10 copies per well. Indeterminate copy number calculations were not used in Fig. 6 since values obtained were below the assay’s lower limit of quantitation.
Gag, Bet and Vif immunoblotting
Cell lysate from FFV-infected CrFK cells or transfected HEK 293T cells were subjected to immunoblot analyses as described [13, 21]. Identical amounts of proteins were separated by SDS-PAGE, blotted, and reacted against different anti-FFV sera. FFV Gag and Bet proteins were detected by rabbit anti-Gag polyclonal serum (1:3000 dilution) and rabbit anti-Bet polyclonal serum (1:2500 dilution) . FIV Vif was detected by a mouse anti-FIV-Vif antibody (NIH AIDS repository, Maryland, USA) at a 1:500 dilution. Membranes were incubated with secondary anti-rabbit polyclonal antibodies or anti-mouse IgG (Sigma, Munich, Germany) conjugated to horseradish peroxidase (1:5000 to 1:2000 dilution) and visualized by chemiluminescence (ECL Western Blot Kit, Amersham Buchler, Braunschweig, Germany). Blots were then probed against actin using mouse anti-actin antibody (1:8000 dilution, Sigma).
GST-capture ELISA for detection of Gag and Bet seroconversion
GST-capture ELISA was performed to detect anti-FFV Gag and anti-FFV Bet antibodies as previously described [55, 74]. Glutathione casein was used to coat 96-well plates (Thermo Fisher Scientific, Waltham, MA) overnight at 4 °C then plates were blocked with casein blocking buffer (0.2% (w/v) casein in PBS and Tween20, Thermo Fisher Scientific). Plates were incubated with BL21 E. coli-produced lysates containing GST-tag, GST-Gag-tag, or GST-Bet-tag recombinant proteins (0.25 µg/µl in casein blocking buffer). Cat sera were pre-adsorbed with GST-tag lysate (2 µg/µl) in a 1:50 dilution and then incubated in duplicate (Fig. 7a, b) or triplicate (Additional file 6) with each GST conjugate. The plates were incubated with anti-cat IgG Protein A peroxidase (1:50,000 dilution, Sigma Aldrich). For the substrate reaction, plates were incubated with TMB substrate before stopping the reaction with sulfuric acid. Absorption (optical density, OD) at 450 nm was measured and the mean reactivity for each was used. Detection cutoff values were determined from negative sera as 2 × (mean + 3 standard deviations). A significant number of reactions at the serum dilution used were out of the linear range of the assay. For anti-Gag antibody titrations, sera from days 28, 42, 70, and 168 p.i. were diluted at 1:100, 1:250, 1:500, 1:1000, 1:2500, and 1:5000. Titer was determined as the highest dilution the cat tested positive for anti-Gag antibodies, using the cutoff formula mentioned above.
FIV Vif antibody capture ELISA
Sera were subjected to an FIV Vif antibody capture ELISA to detect corresponding antibodies in chimeric FFV-Vif-inoculated cats. 96-well plates were coated with 2 ng/µl Vif antigen and incubated overnight at 4 °C. Mouse Vif monoclonal antibody (obtained from Dr. Chris Grant, Custom Monoclonals International, Sacramento, CA) was used as a positive control. After blocking, cat sera (1:100 dilution) or Vif monoclonal antibody (10 ng/µL) were applied in duplicates, then goat anti-cat (or anti-mouse) IgG-HRP (MP Biomedicals, Santa Ana, CA) was used as secondary antibody (1:1000 dilution). TMB reagent was used for the substrate reaction then stopped with sulfuric acid before measuring absorption (450 nm). For detection cutoff, the mean negative sera absorbance readout was used in the following formula: mean + (2 × standard deviation). A number of reactions at the serum dilution used were out of the linear range of the assay.
CLF, SH, GW, DSL, DM, JZ performed the experiments and designed the study together with SVW and ML. RT, EM, and XZ provided valuable information, methods and assisted in experiments and sample collection. CLF, SH, CM, SVW and ML analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
We thank Lutz Gissmann (DKFZ) for his interest in this project and his generous support, Dr. Timo Kehl (DKZF) for the development of the FFV anti-gag PCR and providing training for the FFV capture ELISA, Dr. Chris Grant (Custom Monoclonals Intl, Sacramento, CA, USA) and Dr. John Elder (The Scripps Research Institute, La Jolla, CA, USA) for developing and providing mouse monoclonal Vif antigen for the Vif antibody capture ELISA, and Martha McMillan for cell culture training, animal care, and help with regulatory matters.
The authors declare they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
Consent for publication
Ethics approval and consent to participate
Protocol and animal procedures were approved by the CSU Institutional Animal Care and Use Committee (Protocol #: 13-4104A).
SH, DSL and A-MR were supported by DKFZ PhD stipends. GW was supported by a PhD stipend from the China Scholarship Council. CLF was funded by Morris Animal Foundation (Grant Number: D16FE-402) and The National Institutes of Health (NIH T32 Grant Number: 4T32OD010437-15). The funding bodies played no role in the study design, data collection or analysis, or writing of the manuscript.
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